The mammalian CNS contains a diverse group of glial cells that support neurons, modulate neuronal activity, and enable repair following injury. In addition to astrocytes, oligodendrocytes and microglial cells, it is now recognized that the CNS contains a fourth class of glial cell that exhibits unique properties. These abundant glial cells express the alpha receptor for platelet derived growth factor (PDGF1R), and the chondroitin sulfate proteoglycan NG2, and have been termed "NG2+ glial cells (NG2 cells)". NG2 cells extend highly branched processes decorated with small filopodia-like protrusions, and are evenly distributed throughout gray and white matter regions of the adult CNS in a highly ordered or "tiled" manner, similar to astrocytes and microglial cells. As these cells serve as progenitors to oligodendrocytes, the organization and ongoing dynamics of NG2 cells may be crucial for replacing oligodendrocytes lost through normal aging or following acute demyelination in diseases such as multiple sclerosis. However, the cellular dynamics of NG2 cells have not been studied in the mammalian CNS in vivo, and little is known about the factors that regulate their morphology, distribution, and proliferation of these cells in the adult CNS. To monitor the dynamic behavior of NG2 cells in the adult brain, we recently developed transgenic mice (NG2-lck-EGFP mice) that allow the visualization of NG2 cells in vivo. Using these mice, NG2 cells in the cortex of living mice can be observed during resting-states or after injury, indicating that this approach can be used to help investigate the mechanisms regulating NG2 cell process dynamics, proliferation, and differentiation in the adult brain. Here, I propose to use two-photon imaging of NG2 cells in the somatosensory cortex to study the behavior of NG2 cells within the adult mouse brain. I will perform time-lapse imaging over a period of hours, days and weeks to define the structural dynamics of NG2 cell processes, and to determine the role of cell-cell contact in the regulation of their morphology and proliferation. NG2 cells express ionotropic glutamate receptors, and are unique among glial cells in that they form direct synapses with neurons, providing a means by which neurons could regulate their behavior. To evaluate whether NMDA receptor signaling regulates the dynamic behavior of NG2 cells and their response to focal injury, I will perform similar experiments in transgenic mice in which NMDA receptors have been selectively deleted from NG2 cells. By monitoring the dynamic behavior of NG2 cells in vivo in the normal and injured brain, these studies will further our understanding of the mechanisms that guide the behavior of these ubiquitous progenitors. As these cells have the capacity to replace oligodendrocytes and contribute to the formation of glial scars, these studies may help identify new approaches for accelerating oligodendrocyte regeneration and myelin repair, as well as new strategies for enhancing recovery from diverse CNS injuries. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand the mechanisms that regulate the dynamic behavior of NG2 cells in the adult brain. As NG2 cells have the ability to replace oligodendrocytes and contribute to the formation of glial scars, information gained from these studies may lead to the development of new strategies to promote rapid repair of myelin sheaths in diseases such as multiple sclerosis, and accelerate recovery following CNS injuries such as stroke.